Yutaka Arimura

Protein Tyrosine Phosphatases in Autoimmunity

Protein tyrosine phosphatases (PTPs) are important regulators of many cellular functions and a growing number of PTPs have been implicated in human disease conditions, such as developmental defects, neoplastic disorders, and immunodeficiency. Here, we review the involvement of PTPs in human autoimmunity. The leading examples include the allelic variant of the lymphoid tyrosine phosphatase (PTPN22), which is associated with multiple autoimmune diseases, and mutations that affect the exon-intron splicing of CD45 (PTPRC). We also find it likely that additional PTPs are involved in susceptibility to autoimmune and inflammatory diseases. Finally, we discuss the possibility that PTPs regulating the immune system may serve as therapeutic targets.

PTEN regulation by Akt-EGR1-ARF-PTEN axis.

The PTEN tumour suppressor gene is induced by the early growth response 1 (EGR1) transcription factor, which also transactivates p53, p73, and p300/CBP as well as other proapoptotic and anti‐cancer genes. Here, we describe a novel Akt–EGR1–alternate reading frame (ARF)–PTEN axis, in which PTEN activation in vivo requires p14ARF‐mediated sumoylation of EGR1. This modification is dependent on the phosphorylation of EGR1 at S350 and T309 by Akt, which promotes interaction of EGR1 with ARF at K272 in its repressor domain by the ARF/Ubc9/SUMO system. EGR1 sumoylation is decreased by ARF reduction, and no EGR1 sumoylation is detected in ARF−/− mice, which also exhibit reduced amounts of PTEN. Our model predicts that perturbation of any of the clinically important tumour suppressors, PTEN, EGR1, and ARF, will cause some degree of dysfunction of the others. These results also explain the known negative feedback regulation by PTEN on its own synthesis through PI3 kinase inhibition.

Src Homology Region 2 (SH2) Domain-Containing Phosphatase-1 Dephosphorylates B Cell Linker Protein/SH2 Domain Leukocyte Protein of 65 kDa and Selectively Regulates c-Jun NH2-Terminal Kinase Activation in B Cells

Src homology region 2 (SH2) domain-containing phosphatase-1 (SHP-1) is a cytosolic protein tyrosine phosphatase containing two SH2 domains in its NH2 terminus. That immunological abnormalities of the motheaten and viable motheaten mice are caused by mutations in the gene encoding SHP-1 indicates that SHP-1 plays important roles in lymphocyte differentiation, proliferation, and activation. To elucidate molecular mechanisms by which SHP-1 regulates BCR-mediated signal transduction, we determined SHP-1 substrates in B cells using the substrate-trapping approach. When the phosphatase activity-deficient form of SHP-1, in which the catalytic center cysteine (C453) was replaced with serine (SHP-1-C/S), was introduced in WEHI-231 cells, tyrosine phosphorylation of a protein of about 70 kDa was strongly enhanced. Immunoprecipitation and Western blot analyses revealed that this protein is the B cell linker protein (BLNK), also named SH2 domain leukocyte protein of 65 kDa, and that upon tyrosine phosphorylation BLNK binds to SHP-1-C/S in vitro. In vitro kinase assays demonstrated that hyperphosphorylation of BLNK in SHP-1-C/S-expressing cells was not due to enhanced activity of Lyn or Syk. Furthermore, BCR-induced activation of c-Jun NH2-terminal kinase was shown to be significantly enhanced in SHP-1-C/S transfectants. Taken collectively, our results suggest that BLNK is a physiological substrate of SHP-1 in B cells and that SHP-1 selectively regulates c-Jun NH2-terminal kinase activation.

Comprehensive Expression Profiles of Genes for Protein Tyrosine Phosphatases in Immune Cells

Gene expression analysis suggests that protein tyrosine phosphatases exhibit immune cell–specific expression profiles. Phosphatase Specificity Phosphorylation of proteins is a posttranslational modification that has important functional consequences in many signaling pathways. Thus, regulation of the expression and function of the kinases and phosphatases that control the extent of phosphorylation of target proteins is tightly controlled. The amount of information available about the expression profiles of phosphatase-encoding genes is relatively little compared to that of genes that encode kinases. Arimura and Yagi used data in the RefDIC database to analyze the expression of genes encoding protein tyrosine phosphatases (PTPs) in various immune cells in the mouse. In addition to identifying PTP-encoding genes whose expression was enriched in immune relative to nonimmune tissues, the authors also identified genes whose expression was specific to a given immune cell type. Further study will determine whether these genes might serve as lineage-specific markers of these cells. The phosphorylation and dephosphorylation of signaling molecules play a crucial role in various cellular processes, including immune responses. To date, the global expression profile of protein tyrosine phosphatases (PTPs) in various immune cells has not been described. With the RefDIC (Reference Genomics Database of Immune Cells) database compiled by RIKEN (Rikagaku Kenkyusho), we examined the expression patterns of PTP-encoding genes in mice and identified between 57 and 64 PTP-encoding genes (depending on cutoff values) that were commonly expressed in immune cells. Cells of different lineages contained additional, unique PTP-encoding genes, which resulted in a total of 58 to 76 genes. Compared with cells from nonimmune tissues, immune cells exhibited enhanced expression of the genes encoding 8 PTP-encoding genes, including Ptprc, Ptpn6, and Ptpn22, but had barely detectable expression of 11 PTP-encoding genes, including Ptprd and Tns1. Each immune cell lineage had between 2 and 18 PTP-encoding genes expressed at relatively high or low extents relative to the average expression among immune cells; for example, Ptprj in B cells, Dusp3 in macrophages, Ptpro in dendritic cells, and Ptprg in mast cells. These PTPs potentially play important roles in each cell lineage, and our analysis provides insight for future functional studies.

Multidentate small-molecule inhibitors of vaccinia H1-related (VHR) phosphatase decrease proliferation of cervix cancer cells.

Loss of VHR phosphatase causes cell cycle arrest in HeLa carcinoma cells, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells. We recently reported that VHR is upregulated in several cervix cancer cell lines as well as in carcinomas of the uterine cervix. Here we report the development of multidentate small-molecule inhibitors of VHR that inhibit its enzymatic activity at nanomolar concentrations and exhibit antiproliferative effects on cervix cancer cells. Chemical library screening was used to identify hit compounds, which were further prioritized in profiling and kinetic experiments. SAR analysis was applied in the search for analogs with improved potency and selectivity, resulting in the discovery of novel inhibitors that are able to interact with both the phosphate-binding pocket and several distinct hydrophobic regions within VHRs active site. This multidentate binding mode was confirmed by X-ray crystallography. The inhibitors decreased the proliferation of cervix cancer cells, while growth of primary normal keratinocytes was not affected. These compounds may be a starting point to develop drugs for the treatment of cervical cancer.

Regulatory Roles of IL-2 and IL-4 in H4/Inducible Costimulator Expression on Activated CD4+ T Cells During Th Cell Development

We found a tight correlation among the levels of H4/inducible costimulator (ICOS) expression, IL-4 production, and GATA-3 induction, using activated CD4+ T cells obtained from six different murine strains. BALB/c-activated CD4+ T cells expressed ∼10-fold more H4/ICOS on their surfaces and produced ∼10-fold more IL-4 upon restimulation than C57BL/6-activated CD4+ T cells. BALB/c naive CD4+ T cells were shown to produce much higher amounts of IL-2 and IL-4 upon primary stimulation than C57BL/6 naive CD4+ T cells. Neutralization of IL-4 with mAbs in culture of BALB/c naive CD4+ T cells strongly down-regulated both H4/ICOS expression on activated CD4+ T cells and IL-4 production upon subsequent restimulation. Conversely, exogenous IL-4 added to the culture of BALB/c or C57BL/6 naive CD4+ T cells up-regulated H4/ICOS expression and IL-4 production upon restimulation. In addition, retroviral expression of GATA-3 during the stimulation of naive CD4+ T cells from C57BL/6 or IL-4−/− mice increased H4/ICOS expression on activated CD4+ T cells. A similar effect of IL-2 in the primary culture of BALB/c naive CD4+ T cells appeared to be mediated by IL-4, the production of which was regulated by IL-2. These data suggest that IL-4 induced by IL-2 is critical to the maintenance of high H4/ICOS expression on BALB/c-activated CD4+ T cells.

A Weak Lck Tail Bite Is Necessary for Lck Function in T Cell Antigen Receptor Signaling

Src family kinases are suppressed by a “tail bite” mechanism, in which the binding of a phosphorylated tyrosine in the C terminus of the protein to the Src homology (SH) 2 domain in the N-terminal half of the protein forces the catalytic domain into an inactive conformation stabilized by an additional SH3 interaction. In addition to this intramolecular suppressive function, the SH2 domain also mediates intermolecular interactions, which are crucial for T cell antigen receptor (TCR) signaling. To better understand the relative importance of these two opposite functions of the SH2 domain of the Src family kinase Lck in TCR signaling, we created three mutants of Lck in which the intramolecular binding of the C terminus to the SH2 domain was strengthened. The mutants differed from wild-type Lck only in one to three amino acid residues following the negative regulatory tyrosine 505, which was normally phosphorylated by Csk and dephosphorylated by CD45 in the mutants. In the Lck-negative JCaM1 cell line, the Lck mutants had a much reduced ability to transduce signals from the TCR in a manner that directly correlated with SH2-Tyr(P)505 affinity. The mutant with the strongest tail bite was completely unable to support any ZAP-70 phosphorylation, mitogen-activated protein kinase activation, or downstream gene activation in response to TCR ligation, whereas other mutants had intermediate abilities. Lipid raft targeting was not affected. We conclude that Lck is regulated by a weak tail bite to allow for its activation and service in TCR signaling, perhaps through a competitive SH2 engagement mechanism.

Heterogeneity of HLA-G Genes Identified by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP)

A genomic HLA‐G clone named 7.0E was isolated from a Japanese placenta. The deduced amino acid sequence of the 7.0E was identical to two HLA‐G genomic clones and two cDNA clones previously described. The DNA sequences of α1 and α2 domains of the HLA‐G gene from 5 cell lines also encoded the same amino acids. However, a 14 bp insertion, ATTTGTTCATGCCT, was present in the 3′ untranslated region of 7.0E compared with the originally described HLA‐G clone (HLA 6.0). Polymerase chain reaction (PCR)/single strand conformational polymorphism (SSCP) analysis of exon 8 allowed the HLA‐G gene to be classified into two alternative types, G6.0 and 7.0 E, those correlated to the absence or the presence of the 14 bp stretch. Each group had minor sequence variant(s), and the alleles of the 7.0E‐type were more heterogeneous than those of the G6.0‐ type. The 14 bp deletion is present only in the G6.0‐type of HLA‐G alleles among HLA class I genes. Thus it was suggested that G6.0 alleles were generated after diversification of the HLA‐G.

TCR-induced downregulation of protein tyrosine phosphatase PEST augments secondary T cell responses

We report that the protein tyrosine phosphatase PTP-PEST is expressed in resting human and mouse CD4(+) and CD8(+) T cells, but not in Jurkat T leukemia cells, and that PTP-PEST protein, but not mRNA, was dramatically downregulated in CD4(+) and CD8(+) primary human T cells upon T cell activation. This was also true in mouse CD4(+) T cells, but less striking in mouse CD8(+) T cells. PTP-PEST reintroduced into Jurkat at levels similar to those in primary human T cells, was a potent inhibitor of TCR-induced transactivation of reporter genes driven by NFAT/AP-1 and NF-kappaB elements and by the entire IL-2 gene promoter. Introduction of PTP-PEST into previously activated primary human T cells also reduced subsequent IL-2 production by these cells in response to TCR and CD28 stimulation. The inhibitory effect of PTP-PEST was associated with dephosphorylation the Lck kinase at its activation loop site (Y394), reduced early TCR-induced tyrosine phosphorylation, reduced ZAP-70 phosphorylation and inhibition of MAP kinase activation. We propose that PTP-PEST tempers T cell activation by dephosphorylating TCR-proximal signaling molecules, such as Lck, and that down-regulation of PTP-PEST may be a reason for the increased response to TCR triggering of previously activated T cells.

Normal TCR signal transduction in mice that lack catalytically active PTPN3 protein tyrosine phosphatase

PTPN3 (PTPH1) is a cytoskeletal protein tyrosine phosphatase that has been implicated as a negative regulator of early TCR signal transduction and T cell activation. To determine whether PTPN3 functions as a physiological negative regulator of TCR signaling in primary T cells, we generated gene-trapped and gene-targeted mouse strains that lack expression of catalytically active PTPN3. PTPN3 phosphatase-negative mice were born in expected Mendelian ratios and exhibited normal growth and development. Furthermore, numbers and ratios of T cells in primary and secondary lymphoid organs were unaffected by the PTPN3 mutations and there were no signs of spontaneous T cell activation in the mutant mice with increasing age. TCR-induced signal transduction, cytokine production, and proliferation was normal in PTPN3 phosphatase-negative mice. This was observed using both quiescent T cells and recently stimulated T cells where expression of PTPN3 is substantially up-regulated. We conclude, therefore, that the phosphatase activity of PTPN3 is dispensable for negative regulation of TCR signal transduction and T cell activation.